The molecular nature of regulatory molecules responsible for stimulating early phases of ovarian folliculogenesis (i.e. the growth and differentiation of primordial follicles to primary, secondary, and pre-antral follicles) has been poorly understood. On the other hand, follicle-stimulating hormone (FSH) and luteinising hormone are glycoprotein hormones derived from the pituitary and have generally been accepted as the key factors regulating the later stages of ovarian folliculogenesis.
Moreover FSH is accepted as the single most important factor for stimulating a greater than normal number of follicles to ovulate, a fact that is well illustrated by the wide use of commercial FSH preparations in ovarian hyperstimulation regimes both in medicine and veterinary medicine. Recent studies have indicated that early folliculogenesis is controlled by intraovarian factors of which the granulosa cell-derived stem cell factor (or c-kit ligand) and the oocyte-derived growth differentiation factor-9 (GDF-9) have gained most attention, because both appear to be essential for early mammalian folliculogenesis.
GDF-9 was first described in 1993 as a novel member of the transforming growth factor beta (TGF-β) superfamily which is specifically expressed in the ovary (McPherron and Lee, 1993). Like other members of the TGF-β family, GDF-9 is encoded as a prepropeptide consisting of a signal peptide, a proregion, and a so called C-terminal mature region, which is cleaved from the precursor peptide by an intracellular protease belonging to a group of furin-like proteases. Growth factors of the TGF-β family are characterised by a common pattern of cysteine residues found in the mature region that is likely to form in all members of the family; this is a rigid intramolecular structure known as the “cysteine knot” which is composed of six Cys residues forming three characteristic disulphide bridges in a monomer of a TGF-β family member (Daopin et al., 1992; Schlunegger and Grutter, 1992 and 1993; Griffith et al., 1996; Scheufler et al., 1999). Most members of the TGF-β family have a conserved seventh Cys residue that is responsible for the covalent homodimerisation of two identical monomers (homodimers) or for the heterodimerisation of a given TGF-β family member with another distinct member of the family (heterodimers).
In mice, GDF-9 is expressed in oocytes from the primary stage of follicular development until ovulation (McGrath et al., 1995; Laitinen et al., 1998). Using the mouse GDF-9 sequence as a test sequence for the data base searches we identified, a GDF-9 like expressed sequence tag (EST) cDNA derived from a 2-cell mouse embryo library (Laitinen et al., 1998). We showed that the transcript of this novel factor, GDF-9B, which is 55% homologous to GDF-9, is expressed in oocytes of the mouse ovary at the same time as GDF-9 (Laitinen et al., 1998). Using PCR and primers derived from the mouse EST sequence we amplified a fragment of the corresponding gene from human genomic DNA, mapped the gene locus to chromosome Xp11.2, and deduced the human GDF-9B gene structure from isolated cosmid clones (Aaltonen et al., 1999). Interestingly, in the human ovary, GDF-9 mRNA expression begins in primary follicles slightly earlier than that of GDF-9B (Aaltonen et al., 1999). The mouse and human GDF-9B genes have been cloned and the protein encoded by the gene has also been named bone morphogenetic protein 15 (BMP-15) (Dube et al., 1998).
GDF-9 appears to be essential for ovarian folliculogenesis. From the literature it is known that GDF-9 deficient mice (GDF-9−/−) are infertile due to an early arrest in folliculogenesis (Dong et al., 1996). In GDF-9−/− ovaries folliculogenesis stops at the primary follicle stage when one layer of cuboidal granulosa cells surrounds the oocyte. Even though the oocyte continues to grow, the granulosa cells fail to proliferate and no thecal cell differentiation is associated with follicular enlargement.
The Inverdale fecundity gene (FecX1) was identified as a major gene affecting the prolificacy of a Romney flock of sheep (Davis et al., 1991). Segregation analyses determined that the gene is carried on the X-chromosome, and that ewes carrying a single copy of the gene (I/+) have litter size about 0.6 lambs larger than noncarrier ewes (+/+). The increase in number of lambs born is directly linked to an altered pattern of follicular development and an increase in ovulation-rate above that in the 2 wild-type of ˜1.0 (Shackell et al., 1993; Davis et al., 1991). By contrast, homozygous carrier ewes having two copies of the gene (I/I) are infertile; due to an ovarian failure condition (Davis et al., 1992). In ovaries of the (I/I) ewe, folliculogenesis stops at the primary follicular stage and the phenotype is not dissimilar to that seen in GDF-9 (−/−) mice (Braw-Tal et al., 1993; McNatty et al., 1995; Smith et al., 1997).
A second prolific Romney flock (Hanna, 1995) with no known connection to the Inverdale flock was also shown to carry an X-linked mutation with similar phenotype to Inverdale. Evidence that the Hanna animals carried a mutation (FecXH) in the same gene as for Inverdale was obtained when infertile females were produced by mating Inverdale carrier rams with carrier Hanna ewes (Davis et al., 1995). The Hanna line has been maintained at the Invermay AgResearch Centre as a distinct group alongside the Inverdale line.
In New Zealand Patent Application No. 500844 we, the present inventors, identified in Inverdale sheep a nucleotide substitution beyond the mature peptide processing site of the GDF-913 gene which converts the codon GTC (amino acid valine (V) to GAC (amino acid aspartic acid (D)). We also showed that in Hanna sheep the C nucleotide beyond the mature peptide processing site is converted to a T. This converts a codon CAG (coding for glutamine (Q)) to a codon TAG (coding for termination) thereby resulting in a truncated mature protein. These respective mutations in Inverdale and Hanna are suggested to be the underlying causes for the “streak” ovaries and anovulatory conditions in homozygous Inverdale or Hanna ewes and in the Inverdale cross Hanna ewes.
Previously it has been shown that GDF-9−/− mice are infertile showing that GDF-9 is important for normal fertility in some mammals. However, with the discovery of the related oocyte-specific factor GDF-9B several novel findings were found by us to support the notion that: (1) GDF-9B is essential for normal folliculogenesis in some mammals; (2) that GDF-9B is critically important for determining the ovulation rate in some mammals and; (3) since GDF-9 and GDF-9B are coexpressed by oocytes, they function co-operatively to enhance both follicular development and ovulation rate. Collectively these novel hypotheses were only made possible by our discovery of the Inverdale and Hanna GDF-9B mutations in sheep.
The inventors have for the first time determined the full gene structure of the sheep GDF-9B gene encoding the wild-type protein and have shown that it is necessary for maintaining normal ovarian folliculogenesis in sheep. The inventors have also identified the full gene structure of the GDF-9B variants in Inverdale and Hanna sheep which cause higher than normal ovulation rates in heterozygous animals and infertility in homozygous animals. It is broadly to the full-length wildtype and mutated GDF-9B sequences and variants thereof and their use in the modulation of mammalian fertility that the present invention is directed.
All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in New Zealand or in any other country.